- Total grants
- Total funders
- Total recipients
- Earliest award date
- 17 Oct 2005
- Latest award date
- 30 Sep 2017
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
The aim of this project is to develop a completely xeno-free culture system for human mesenchymal stem cells (hMSCs), cultured on synthetic peptide hydrogels (PeptieGel Design, Ltd.) and ‘mechano-cultured’ in a simple wave tank bioreactor with dynamic hydrostatic pressure acting as a dominant stimulus to control cell growth and differentiation into active osteoblasts and chondrocytes. Our hypothesis is that the changing pressure (which replicates the loading experienced during anabolic exercise) will stimulate mechanotransduction pathways in the cell that promote the growth, asymmetric division and subsequent differentiation of hMSCs by enhancing the signal transduction of autocrine and paracrine growth and transcription factors. Our project goals therefore are to: Combine a simple wave tank bioreactor with our hydrostatic pressurisation system. Culture hMSCs in synthetic peptide hydrogels as encapsulated 3D microcarrier beads. Compare growth of conventionally cultured (T-flasks) hMSCs to the bioreactor cultured hMSCs Switch to differentiation media (chondrogenic and osteogenic) to determine how hMSCs differentiate during the end stages of mechanoculture. A summary of these goals is attached in the supporting document (fig. 1.)
In vitro characterisation of neurons and glia derived from mouse ES cells having Y chromosome gene deficiencies 27 Apr 2017
The brain is a sexually dimorphic organ, which can explain why many psychiatric disorders are gender-biased. While it has been thought that fetal sex hormones play a crutial role in brain sexual differentiation, emerging evidence indicates that genetic and/or epigenetic factors encoded by sex chromosome genes are also involved. Here, we focus on several Y-linked genes expressed in the male brain, which may act in a dominant manner. Sry, Uty and Smcy genes, whose products can participate in histone modification, are of particular interest. However, their functions in the brain remain unknown mainly because of difficulties in conventional gene-targeting of Y loci in embryonic stem cells (ESCs). In this project, we will take advantage of Crispr/Cas genome-editing technology that allows us to modify Y-linked genes. Mutant alleles of these three genes in ESCs were created and then differentiation will be induced of neurons and glia from the mutant ES cells. Differential gene expression between wild-type and mutant cells will be analysed by RT-RCR and bisulfite PCR, and subsequently epigenetic status of these differential genes will be elucidated. The project will reveal influences of Y-linked genes on sex-specific epigenetic modification and lead to better understanding of gender-biased psychiatric disorders.
The role of inheritance and the EGF pathway in mis-segregation of chromosomes during mitosis. 27 Apr 2017
I am proposing to research how the mis-segregation of whole chromosomes during mitosis impacts future cell divisions and how this is coupled to spindle assembly pathways and epidermal growth factor (EGF) signaling. During this project I will investigate whether a gain or loss of a chromosome, due to mis-segregation, predisposes daughter and grand-daughter cells to further segregation errors. Additionally, I will research whether the inhibition of EGF signaling will exacerbate chromosomal mis-segregation. The research will be carried out on non-small cell lung cancer (NSCLC) cell lines, which will be transfected with GFP-CENP-A, marking kinetochores, or GFP-tubulin, marking the spindle, and treated with SiR-DNA, allowing visualisation of chromosomes. These fluorescent reporter constructs will allow chromosomal mis-segregation and spindle geometry to be observed during live cell imaging. The live-cell imaging experiments will then be repeated in the presence of Gefitinib, allowing observations to be made whilst EGF signaling is inhibited. The primary goals of this research are firstly being able to provide key insight into how the EGF pathway regulates chromosomal instability (CIN) and cell fates in NSCLC, and secondly to be able to establish how the mis-segregation of whole chromosomes impacts future cell divisions.
Protein O-GlcNAcylation is an essential posttranslational modification regulated by two opposing highly conserved enzymes, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), influencing a wide range of biological processes. The dysregulation of the O-GlcNAc system is linked to neurodegeneration, cancer and diabetes. OGT consists of a catalytic domain, and an N-terminal domain comprising 13.5 tetratricopeptide repeats (TPRs). This TPR domain is believed to mediate protein-protein interactions. The OGT gene is located on the Xq13.1 band. Four XLID causing mutations were identified in the OGT gene so far. Residing in the TPR region, they lead to a decrease of steady-state global OGT protein levels and effects on the O-GlcNAc proteome. In my summer research project, I would like to focus on the A319T (c.1193G>A) missense mutation. Having been found in a French family with three XLID affected sons, this mutation is regarded as the possible pathogenic factor in this particular case. My key goals include the production of the A319T mutated OGT in E. coli, to be further on purified and characterised in vitro. The structural integrity will be checked and assays screening the enzymatic activity will be carried out. I hope to detect differences in enzyme kinetics caused by the structural changes.
Investigating the association between genetic and epigenetic variability in immune genes and depression in young adults. 27 Apr 2017
Depression is one of the most commonly occurring mental health disorder in student populations, and the general population. However, despite its prevalence the environmental and biological factors that cause depression, and the underlying pathophysiology of the disorder remain poorly understood. The current diagnosis of depression is largely subjective, and there are no clinically validated biomarkers that can be used to assist in diagnosis and treatment selection. Variability in genes encoding inflammatory cytokines has been linked to the aetiology of depression. The aim of this project is to identify candidate inflammatory biomarkers which could potentially be useful for diagnosis and treatment selection in depression. The main objectives will be to determine whether polymorphisms and DNA methylation patterns in the interleukin 1 beta (IL-1B) and Tumour Necrosis Factor alpha genes are associated with depression in the student population in Northern Ireland.
Developing an in vivo MT nucleation assay to investigate g-tubulin independent centrosomal MT nucleation 27 Apr 2017
Centrosomes are major microtubule organising centres (MTOCs) in animal cells. During mitosis they recruit large numbers of gamma-tubulin ring complexes (g-TuRCs), which nucleate and anchor the microtubules required for spindle formation. Recent work in the Conduit lab has surprisingly shown that centrosomes lacking g-TuRCs can still organise microtubules. Nevertheless, it remains unclear if these microtubules are generated at centrosomes, or generated in the cytoplasm and then anchored at centrosomes. I aim to establish an in vivo microtubule nucleation assay to test these alternative possibilities. Drosophila larval brains, which are highly mitotically active, will be dissected from either wild-type flies or from mutant flies where the centrosomes lack g-TuRCs. They will be cooled on ice for 40 minutes in order to depolymerise all microtubules and then transferred to 25 degrees and chemically fixed at different timepoints. The brains will be stained for microtubules, centrosomes and mitotic DNA using antibodies already available in the Conduit lab and images will be taken on a confocal microscope. The location and intensity of new microtubule growth will be assessed. If the g-TuRC negative centrosomes do nucleate microtubules, the assay will be used to test candidate proteins for their role in centrosomal non-g-TuRC mediated microtubule nucleation.
Integrative and conjugative elements (ICEs) are mobile genetic elements present in both gram-positive and gram-negative bacteria. They mostly reside in the host chromosome and under certain conditions, will excise and transfer to a new host via the conjugation machinery. ICEs have been found to provide the host with a wide range of phenotypes, including antibiotic and heavy metal resistance and the ability to colonise a eukaryotic host, promote virulence and biofilm formation. The ability of ICE to spread to different species of bacteria through horizontal gene transfer is a major factor in bacterial evolution. Bioinformatics approaches have been increasingly used to identify possible ICEs through sequence similarity. In this project, we aim to find out the effectiveness of using an algorithm, DLIGHT (Distance Likelihood based Inference of Genes Horizontally Transferred) that was originally used to detect lateral gene transfer, to identify integrative and conjugative elements. We will achieve this by assessing DLIGHT's ability to recover already documented ICEs. We will also use DLIGHT to test certain sequences which we suspect to contain ICEs. The predictions of new ICEs will then be vetted through manual analysis and collaboration with experimentalists.
Immunomodulatory effect of cigarette- and electronic cigarette- exposed respiratory pathogens on macrophages in vitro 27 Apr 2017
It has been established that cigarette smoke and E-cigarette vapour can activate inflammatory response and impair antimicrobial functions of human immune cells. However, the direct effects of cigarette smoke and E-cig vapour on respiratory pathogens is less well known. The aim of this project is to further explore the effect of cigarette smoke extract (CSE) and e-cigarette smoke extract (ECSE) on important human respiratory pathogens and their interaction with the host. The immunomodulatory effects of CSE/ECSE exposed respiratory pathogens have on macrophages in vitro will be determined. Western blot analysis will be used to detect activation of the canonical NF-kappaB pathway and caspase-1 activation whilst ELISA assays will be utilized to measure the expression of the cytokines IL-8, IL-12, TNF-alpha and IL-1beta. In addition, susceptibility of bacteria to macrophage phagocytosis and killing will be measured. The findings of this project will provide valuable information about the direct effect of cigarette smoke and e-cigarette on the lung microbiome and its interaction with host immune cells.
The response of the gut microbiota to a range of anti-diabetic medications and its impact on glucose control 27 Apr 2017
The key goal of this project is investigating the effect dietary intake and gut microbiota have on glucose regulation in established diabetic patients prescribed a range of anti-diabetic medications. Evidence from the literature suggests an altered microbiota is associated with changes in multiple heath parameters, including glucose regulation (Cani et al. 2014). Previous studies have illustrated that dietary intake impacts the gut microbiota profile and can enhance its diversity, an independent marker of improved functionality in vivo (Cotillard et al. 2013). This project involves microbial DNA extraction to study the effect of various anti-diabetic medications on the gut microbiota profile (from time of prescribing to 8 week follow up). Dietary intake will be assessed to determine the role of diet on the baseline microbiota in diabetic candidates. Dr. O’Connor’s team have extensive expertise in analysing gut microbiota profiles and determining the role of diet in shaping microbiota in specific populations (Power et al. 2015, O’Connor et al. 2014, Power et al. 2014, O’Connor et al. 2013), especially during aging and aging-related health loss (Claesson et al. 2012). Dr.O'Connors group have numerous ongoing dietary intervention trials to determine the role of specific food ingredients on gut microbiota in specific population groups.
Glutamate is the major excitatory transmitter in the mammalian CNS and is involved in key physiological functions and in a variety of neurological conditions, including stroke, chronic pain, anxiety and epilepsy. The actions of glutamate are mediated via four classes of receptors: AMPA, Kainate, NMDA and Metabotropic. Kainate receptors (KARs) are composed of five subunits (GluK1-5). KARs are involved in many physiological functions, however they have also been implicated in the pathophysiology of epilepsy. Little is known about the physiological roles of KARs in neurological disorders, compared with the other glutamate receptor classes, mainly due to the lack of selective drugs. Recently this setting started to change with the development of selective GluK1 antagonists. This project aims to test the ability of a novel GluK2 antagonist UBP2002 (produced at the University of Bristol) to prevent the development of and to reverse epileptic activity induced by electrical stimulation on Hippocampal slices. Current antiepileptic drugs (AEDs), although effective in controlling epileptic seizures in the majority of patients, have limited efficacy against drug resistant epilepsies. Therefore, there is a need for new drugs acting on different targets than those of currently available AEDs and for the development of new therapies for epilepsy.
Ageing increases physiological frailty; a failure to respond to intracellular and extracellular challenges. In mammals the entry of cells into replicative senescence is now known to be a primary ageing mechanism. Changes in transcripts encoding mRNA splicing regulators have been shown to be associated with age and replicative senescence both in vitro and in vivo. Specifically, in normal human fibroblast cultures, a dramatic shift in alternative isoform usage occurs which we have shown can be reversed by a number of small molecules based upon resveratrol. However, the causal relationship between entry into the senescent state and this shift in isoform use remains unclear as does the universality of splicing factor shifts between individuals. To address the goals of this summer studentship are: (i) to study changes in splicing factor expression in fibroblasts derived from individuals who show accelerated replicative senescence by different kinetic routes. (ii) to study such changes in isogenic telomerase immortalised controls. (iii) to determine if the changes seen at senescence are reversible with resveratrol based modulators of splicing factor expression.
Many new inhaled medicines fail during development due to the induction of a "foamy" alveolar macrophage response in pre-clinical studies. It is not fully understood if a highly vacuolated macrophage response to an inhaled stimulus is adverse or adaptive. Macrophages dynamically alter their phenotype and function depending on their underlying microenvironment resulting in abnormal shifts in their polarization state between classically (M1) and alternatively (M2) activated. M1 describes the classically activated macrophage which acts as an innate immune effector cell characterised by increased production of pro-inflammatory cytokines. M2 macrophages are alternatively activated, responsible for signalling wound-healing cell secreting anti-inflammatory cytokines. At the university, high-content screens have been developed to assess macrophage cell health and morphometric changes induced by different stimuli. However, the mechanism of these changes has not been explained and it is unknown if macrophage responses are triggered by macrophage polarization. The aim of this study is to investigate macrophage polarization and cellular responses of alveolar-like macrophages derived from the human monocyte cell line U937, to different chemical inducers. Improved understanding of macrophage responses to inhaled stimuli is necessary for further progress in this area, and to fully characterise the macrophage response to elucidate its role in airway pathophysiology.
Human Skeletal Muscle Wound Repair: An Association between Genetic Variation and Stem Cell Migration? 27 Apr 2017
Insufficient recovery following exercise-induced muscle damage (EIMD) can lead to muscle injury, with natural genetic variation perhaps influencing the extent of both. Skeletal muscle regeneration is a complex process that is mediated by muscle stem cells (called satellite cells). However, little is known about whether specific genetic variations (polygenic profile) influence dynamics of satellite cells and their impact on skeletal muscle regeneration. For that reason, we aim to conduct an in vitro muscle cell damaging study on primary skeletal muscle stem cells (previously derived from participants with specific polygenic profiles) to provide potential mechanisms regarding the individual response to injury in vitro. We hypothesise that activated satellite cells from individuals with a specific genetic profile will display better rates of recovery, following mechanical injury, compared with cells from participants without this profile. Our objective is to utilise stem cell-based research to provide oversight of the in-depth genetic mechanisms underlying the adaptations of muscle cells to injury repair. Our ultimate aim is to use current technology to prevent injury and promote recovery initially in the sporting world, but ultimately in the wider population as exercise becomes more widely accepted as a health intervention.
Conventional hPSCs represent a relatively late stage of embryonic development, termed the primed phase of pluripotency. Their use in research and medical applications is problematic because they display a differentiation bias and do not generate all cell lineages efficiently. The Smith laboratory has recently defined culture conditions that capture cells in an earlier phase, termed naïve pluripotency. Naive hPSCs have potential implications for more effective stem cell therapies because they don't display a differentiation bias. Very little is known about the genes that govern human naïve pluripotency in culture. The transcription factor KLF17, which is present in naïve hPSCs but absent in primed hPSCs, is of particular interest because it is specific to primates and not well studied. The key goal of this project is to generate a fluorescent reporter for KLF17. Alternative reporter designs will be trialled by fusing fluorescent proteins to either the start or the end of the endogenous KLF17 protein, in order to achieve optimal fidelity and sensitivity. The reporter(s) will then be exploited to monitor the dynamics of KLF17 expression in live cells both following withdrawal of factors to initiate differentiation, and also during the process of generating naïve cells by resetting.
Search for microRNAs produced by gut bacteria with the potential to regulate host cell gene expression 27 Apr 2017
Through extensive analysis of the genome of gut commensal bacterium Bacteriodes spp., sequences predicted to form short (s)RNA hairpin structures homlogous to human microRNAs, and in particular miR-4787-5p. The target of this miRNA is ZBTB7A, a transcription factor involved in the transcriptional suppression of glycolysis. Prof Carding's group has previously shown that Bacteriodes spp. exports macromolecules including sRNAs in nanoscale microvesicles (outer membrane vesicles; OMV) that can be acquired by other gut bacteria and importantly for this project, intestinal epithelial cells. The aim of the project therefore is to determine if Bacteroides miR-4787-5p is structurally and functionally equivalent to mammalian miRNAs. The first goal of the project is to verify the sequence of the candidate miRNA, miR-4787-5p, through PCR and subsequent sequencing, and determine where in the bacterial cell it is expressed, and if it is present in OMVs. The next goal is to overexpress the miR-4787-5p sequences in a mammalian cell via a mammalian expression vector and determine the effect on expression of ZBTB7A, which is the target of the homologous mammalian miRNA, and the cells' metabolism and growth using moecular and cell based assays already established in the laboratory.
Bovine Respiratory Syncitial Virus Vaccine 27 Apr 2017
Bovine respiratory syncitial virus (BRSV) is a leading cause of enzootic pneumonia in calves and significantly contributes to the Bovine Respiratory Disease Complex found in feedlot cattle. It is proposed to develop a new vaccine using the Semliki Forest Virus (SFV) virus replicon particle (VRP) vaccine platform. The SFV VRP is a promising vaccine platform, due in part to its efficacious immunogenicity in cattle and its ability to express protein to high concentrations in the cells it infects. Furthermore, the replicon vectors are single cycle, propagation-defective particles that are not able to spread beyond the initial infected cells. The BRSV fusion (F) protein is known to be immunogenic thus making it a good candidate protein to target for gene expression. It is proposed to clone the F protein sequence into a VRP expression plasmid in place of the SFV structural proteins. The proposed project will involve generating the VRP plasmids, generation of VRP stocks and confirmation that this VRP can express the F protein. Human respiratory syncitial virus (hRSV), a closely related virus of human importance, also expresses the F protein. Thus BRSV infection in bovines is a relevant model for hRSV, as there is no current efficacious vaccine available.
Investigating the effects of phosphatases on Trk signalling and cell fate in neuroblastoma 27 Apr 2017
Neuroblastoma is highly variable in outcome; it might be aggressive and fatal, but might also spontaneously regress. Expression of TrkA is strongly associated with patient survival (due to differentiation or apoptosis of the cancer cells), whereas TrkB is associated with unfavorable, metastatic cancer. My supervisor's group generated cell lines that over-express the different Trk-receptors. These cell lines mimic the characteristics of the human tumours; i.e., TrkA cells differentiate upon NGF stimulation, and TrkB cells proliferate upon ligand (BDNF) binding. My supervisor's group found that NGF and BDNF activate the same major pathways but with different dynamics (i.e., different signal duration). Therefore, we hypothesise that the different downstream dynamics of TrkA and TrkB signalling is responsible for the diffent cell fates, differentiation and proliferation, respectively. Since phosphatases are important regulators of signal duration; my aim is to investigate the activity of five phosphatases in TrkA and TrkB cells by Western blotting. Moreover, we wold like to test if we can modify cell fate by altering phosphatase activity in TrkB over-expressing neuroblastoma cells (i.e., induce differentiation of cells instead of proliferation).
Prostate cancer (PCa) is the most commonly diagnosed cancer affecting men with one in eight men developing the disease within their lifetime. Androgen receptor (AR) activation plays a critical role in PCa development and progression. Therapies which antagonise AR signalling are initially effective, however the majority of men relapse to develop an incurable form of PCa termed castrate-resistant PCa (CRPC) in which AR activation occurs independently of androgen stimulation. Consequently, there is an urgent need for the development of novel therapeutic strategies. The AR is not solely regulated by its cognate steroid hormone; it is also regulated by a variety of co-regulatory proteins, some of which are responsible for post-translationally modifying the AR. Interestingly, AR phosphorylation has been shown to be important in directly modulating AR activity. However, the protein kinases involved in AR activation in advanced and therapy resistant PCa remain ill defined. In a comprehensive siRNA kinome screen designed to identify kinases involved in the regulation of AR activity, the threonine/serine kinase, IKBKE, was identified as a putative AR activator. Therefore, subsequent evaluation of IKBKE signalling in PCa progression will be investigated. Specifically, this project will determine the effect of IKBKE inhibition on PCa cell growth and migration.
Nitric oxide (NO) is a potent regulator of vascular tone. Until relatively recently, it was assumed that the isoform of NO synthase responsible for tonic NO release was endothelial nitric oxide synthase (eNOS). However, we now know that in humans, neuronal NOS (nNOS) is the primary NOS isoform responsible for regulating vascular tone in vivo. Neuronal NOS is also activated by mental stress and contributes directly to resistance vessel vasodilatation. However, our preliminary data indicate that this response is biphasic, suggesting a second mechanism underlying the vasodilatory response to stress. We hypothesise that this additional mechanism may be mediated through agonism of beta2 adrenoceptors. This hypothesis will be tested in healthy volunteers exposed to mental stress (Stroop test), using the gold-standard technique of venous occlusion plethysmography to measure forearm blood flow, coupled with intra-arterial infusions of selective inhibitors of nNOS and beta2 adrenoceptors. The key goals of this research are (i) to better define the regulation of vascular tone in healthy humans with a view to understanding potential mechanisms underlying vascular dysfunction in disease states; and (ii) gain a broader understanding of early experimental medicine approaches in the clinical setting.
The role of protein kinases in the regulation of Rif1 during DNA damage and spindle damage in eukaryotic cells 27 Apr 2017
Rif1 (Rap1-Interacting Factor 1) protein is a well-conserved DNA-damage-response factor, required for cell survival after DNA damage and for proper execution of DNA replication. Little is known about how does Rif1 achieve these functions or how it is regulated. This project will investigate potential roles for certain protein kinases, in phosphorylating Rif1 in response to telomere and spindle damage. The protein kinases will be selected from a list of factors demonstrated to interact with Rif1 in high throughput assays. The key goal is to Identify the protein(s) phosphorylating Rif1, which may help us understand the mechanisms by which telomere dysfunction is involved in carcinogenesis.